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. 2016 Feb 18;5(5):e1143995. doi: 10.1080/2162402X.2016.1143995

Figure 2.

Figure 2.

De novo ceramide synthesis involved in LMP-mediated cell death initiated by type II CD20 mAb. (A) The inhibition of cell death in Ramos cells by A-SMase, N-SMase and ceramide synthase inhibitors (Imip, 3-OMe-SM and FB1, respectively) was assessed by FCM. Error bars indicate SD (n = 3). *p < 0.05. (B) The exogenous ceramide (C2-Ceramide) induced cell death in both Raji and Ramos cells in a dose-dependent manner. *p < 0.05. (C) Time course study of ceramide generation in B cells induced by CD20 mAbs. The ceramide levels were quantitated as described in “Supplemental Experimental Procedures.” Results are representative of three independent experiments. (D) The generation of ceramide stimulated by 11B8 was inhibited by FB1. Raji cells were treated with FB1 prior to the addition of CD20 mAbs. *p < 0.05. Detection of total lysosomal volume in cells treated with mAbs and FB1. Cells were incubated with CD20 mAbs (10 μg/mL) and FB1 (25 μM). After that, cells were labeled with LysoTracker green and the volume of the lysosomal compartment measured by confocal microscopy (E) and FCM (F) after 4 h. (G and H) The assessment of LMP by evaluating the release of cathepsin B (red) into cytoplasm. Scale bars: 10 μm.